Light-Activated Synthetic Rotary Motors in Lipid Membranes Induce Shape Changes Through Membrane Expansion.

Membranes are the key structures to separate and spatially organize cellular systems. Their rich dynamics and transformations during the cell cycle are orchestrated by specific membrane-targeted molecular machineries, many of which operate through energy dissipation. Likewise, man-made light-activated molecular rotary motors have previously shown drastic effects on cellular systems, but their physical roles on and within lipid membranes remain largely unexplored. Here, the impact of rotary motors on well-defined biological membranes is systematically investigated. Notably, dramatic mechanical transformations are observed in these systems upon motor irradiation, indicative of motor-induced membrane expansion. The influence of several factors on this phenomenon is systematically explored, such as motor concentration and membrane composition., Membrane fluidity is found to play a crucial role in motor-induced deformations, while only minor contributions from local heating and singlet oxygen generation are observed. Most remarkably, the membrane area expansion under the influence of the motors continues as long as irradiation is maintained, and the system stays out-of-equilibrium. Overall, this research contributes to a comprehensive understanding of molecular motors interacting with biological membranes, elucidating the multifaceted factors that govern membrane responses and shape transitions in the presence of these remarkable molecular machines, thereby supporting their future applications in chemical biology.

Measuring Thousands of Single-Vesicle Leakage Events Reveals the Mode of Action of Antimicrobial Peptides.

Host defense or antimicrobial peptides hold promise for providing new pipelines of effective antimicrobial agents. Their activity quantified against model phospholipid membranes is fundamental to a detailed understanding of their structure-activity relationships. However, classical characterization assays often lack the ability to achieve this insight. Leveraging a highly parallelized microfluidic platform for trapping and studying thousands of giant unilamellar vesicles, we conducted quantitative long-term microscopy studies to monitor the membrane-disruptive activity of archetypal antimicrobial peptides with a high spatiotemporal resolution. We described the modes of action of these peptides via measurements of the disruption of the vesicle population under the conditions of continuous peptide dosing using a range of concentrations and related the observed modes to the molecular activity mechanisms of these peptides. The study offers an effective approach for characterizing membrane-targeting antimicrobial agents in a standardized manner and for assigning specific modes of action to the corresponding antimicrobial mechanisms.

An ultrasensitive microfluidic approach reveals correlations between the physico-chemical and biological activity of experimental peptide antibiotics.

Antimicrobial resistance challenges the ability of modern medicine to contain infections. Given the dire need for new antimicrobials, polypeptide antibiotics hold particular promise. These agents hit multiple targets in bacteria starting with their most exposed regions-their membranes. However, suitable approaches to quantify the efficacy of polypeptide antibiotics at the membrane and cellular level have been lacking. Here, we employ two complementary microfluidic platforms to probe the structure-activity relationships of two experimental series of polypeptide antibiotics. We reveal strong correlations between each peptide's physicochemical activity at the membrane level and biological activity at the cellular level. We achieve this knowledge by assaying the membranolytic activities of the compounds on hundreds of individual giant lipid vesicles, and by quantifying phenotypic responses within clonal bacterial populations with single-cell resolution. Our strategy proved capable of detecting differential responses for peptides with single amino acid substitutions between them, and can accelerate the rational design and development of peptide antimicrobials.

A Microfluidic Platform for Sequential Assembly and Separation of Synthetic Cell Models.

Cell-sized vesicles like giant unilamellar vesicles (GUVs) are established as a promising biomimetic model for studying cellular phenomena in isolation. However, the presence of residual components and byproducts, generated during vesicles preparation and manipulation, severely limits the utility of GUVs in applications like synthetic cells. Therefore, with the rapidly growing field of synthetic biology, there is an emergent demand for techniques that can continuously purify cell-like vesicles from diverse residues, while GUVs are being simultaneously synthesized and manipulated. We have developed a microfluidic platform capable of purifying GUVs through stream bifurcation, where a vesicles suspension is partitioned into three fractions: purified GUVs, residual components, and a washing solution. Using our purification approach, we show that giant vesicles can be separated from various residues─which range in size and chemical composition─with a very high efficiency (e = 0.99), based on size and deformability of the filtered objects. In addition, by incorporating the purification module with a microfluidic-based GUV-formation method, octanol-assisted liposome assembly (OLA), we established an integrated production-purification microfluidic unit that sequentially produces, manipulates, and purifies GUVs. We demonstrate the applicability of the integrated device to synthetic biology through sequentially fusing SUVs with freshly prepared GUVs and separating the fused GUVs from extraneous SUVs and oil droplets at the same time.

Standardizing characterization of membrane active peptides with microfluidics.

Antimicrobial peptides (AMPs) are emerging as important players in the fight against antibiotic resistance. In parallel, the field of microfluidics has matured and its benefits are being exploited in applications of biomimetics and standardized testing. Membrane models are essential tools extensively utilized in studying the activity and modes of action of AMPs. Here, we describe how the utilization of microfluidic platforms in characterizing membrane active peptides can develop a reliable colorful image that classical techniques have rendered black and white.

Switching Cytolytic Nanopores into Antimicrobial Fractal Ruptures by a Single Side Chain Mutation.

Disruption of cell membranes is a fundamental host defense response found in virtually all forms of life. The molecular mechanisms vary but generally lead to energetically favored circular nanopores. Here, we report an elaborate fractal rupture pattern induced by a single side-chain mutation in ultrashort (8-11-mers) helical peptides, which otherwise form transmembrane pores. In contrast to known mechanisms, this mode of membrane disruption is restricted to the upper leaflet of the bilayer where it exhibits propagating fronts of peptide-lipid interfaces that are strikingly similar to viscous instabilities in fluid flow. The two distinct disruption modes, pores and fractal patterns, are both strongly antimicrobial, but only the fractal rupture is nonhemolytic. The results offer wide implications for elucidating differential membrane targeting phenomena defined at the nanoscale.

Towards understanding single-channel characteristics of OccK8 purified from Pseudomonas aeruginosa.

Antibiotic resistance in Gram-negative bacteria causes serious health issues worldwide. Bacteria employ several resistance mechanisms to cope with antimicrobials. One of their strategies is to reduce the permeability of antibiotics either through general diffusion porins or substrate-specific channels. In this study, one of the substrate-specific channels from Pseudomonas aeruginosa, OccK8 (also known as OprE), was investigated using single-channel electrophysiology. The study also includes the investigation of permeability properties of several amino acids with different charged groups (i.e. arginine, glycine and glutamic acid) through OccK8. We observed four different conformations of the same OccK8 channel when inserted in lipid bilayers. This is in contrast to previous studies where heterologous expressed OccK8 in E. coli showed only one conformation. We hypothesized that the difference in our study was due to the expression and purification of the native channel from P. aeruginosa. The single-channel uptake characteristics of the porin showed that negatively charged glutamic acid preferentially interacted with the channel while the positively charged arginine molecule showed infrequent interaction with OccK8. The neutral amino acid glycine did not show any interaction at the physiological conditions.

Aerosol-jet printing facilitates the rapid prototyping of microfluidic devices with versatile geometries and precise channel functionalization.

Microfluidics has emerged as a powerful analytical tool for biology and biomedical research, with uses ranging from single-cell phenotyping to drug discovery and medical diagnostics, and only small sample volumes required for testing. The ability to rapidly prototype new designs is hugely beneficial in a research environment, but the high cost, slow turnaround, and wasteful nature of commonly used fabrication techniques, particularly for complex multi-layer geometries, severely impede the development process. In addition, microfluidic channels in most devices currently play a passive role and are typically used to direct flows. The ability to "functionalize" the channels with different materials in precise spatial locations would be a major advantage for a range of applications. This would involve incorporating functional materials directly within the channels that can partake in, guide or facilitate reactions in precisely controlled microenvironments. Here we demonstrate the use of Aerosol Jet Printing (AJP) to rapidly produce bespoke molds for microfluidic devices with a range of different geometries and precise "in-channel" functionalization. We show that such an advanced microscale additive manufacturing method can be used to rapidly design cost-efficient and customized microfluidic devices, with the ability to add functional coatings at specific locations within the microfluidic channels. We demonstrate the functionalization capabilities of our technique by specifically coating a section of a microfluidic channel with polyvinyl alcohol to render it hydrophilic. This versatile microfluidic device prototyping technique will be a powerful aid for biological and bio-medical research in both academic and industrial contexts.

An Integrated Microfluidic Platform for Quantifying Drug Permeation across Biomimetic Vesicle Membranes.

The low membrane permeability of candidate drug molecules is a major challenge in drug development, and insufficient permeability is one reason for the failure of antibiotic treatment against bacteria. Quantifying drug transport across specific pathways in living systems is challenging because one typically lacks knowledge of the exact lipidome and proteome of the individual cells under investigation. Here, we quantify drug permeability across biomimetic liposome membranes, with comprehensive control over membrane composition. We integrate the microfluidic octanol-assisted liposome assembly platform with an optofluidic transport assay to create a complete microfluidic total analysis system for quantifying drug permeability. Our system enables us to form liposomes with charged lipids mimicking the negative charge of bacterial membranes at physiological pH and salt concentrations, which proved difficult with previous liposome formation techniques. Furthermore, the microfluidic technique yields an order of magnitude more liposomes per experiment than previous assays. We demonstrate the feasibility of the assay by determining the permeability coefficient of norfloxacin and ciprofloxacin across biomimetic liposomes.

Indole Pulse Signalling Regulates the Cytoplasmic pH of E. coli in a Memory-Like Manner.

Bacterial cells are critically dependent upon pH regulation. Here we demonstrate that indole plays a critical role in the regulation of the cytoplasmic pH of Escherichia coli. Indole is an aromatic molecule with diverse signalling roles. Two modes of indole signalling have been described: persistent and pulse signalling. The latter is illustrated by the brief but intense elevation of intracellular indole during stationary phase entry. We show that under conditions permitting indole production, cells maintain their cytoplasmic pH at 7.2. In contrast, under conditions where no indole is produced, the cytoplasmic pH is near 7.8. We demonstrate that pH regulation results from pulse, rather than persistent, indole signalling. Furthermore, we illustrate that the relevant property of indole in this context is its ability to conduct protons across the cytoplasmic membrane. Additionally, we show that the effect of the indole pulse that occurs normally during stationary phase entry in rich medium remains as a "memory" to maintain the cytoplasmic pH until entry into the next stationary phase. The indole-mediated reduction in cytoplasmic pH may explain why indole provides E. coli with a degree of protection against stresses, including some bactericidal antibiotics.

A synthetic enzyme built from DNA flips 107 lipids per second in biological membranes.

Mimicking enzyme function and increasing performance of naturally evolved proteins is one of the most challenging and intriguing aims of nanoscience. Here, we employ DNA nanotechnology to design a synthetic enzyme that substantially outperforms its biological archetypes. Consisting of only eight strands, our DNA nanostructure spontaneously inserts into biological membranes by forming a toroidal pore that connects the membrane's inner and outer leaflets. The membrane insertion catalyzes spontaneous transport of lipid molecules between the bilayer leaflets, rapidly equilibrating the lipid composition. Through a combination of microscopic simulations and fluorescence microscopy we find the lipid transport rate catalyzed by the DNA nanostructure exceeds 107 molecules per second, which is three orders of magnitude higher than the rate of lipid transport catalyzed by biological enzymes. Furthermore, we show that our DNA-based enzyme can control the composition of human cell membranes, which opens new avenues for applications of membrane-interacting DNA systems in medicine.

Direct Optofluidic Measurement of the Lipid Permeability of Fluoroquinolones.

Quantifying drug permeability across lipid membranes is crucial for drug development. In addition, reduced membrane permeability is a leading cause of antibiotic resistance in bacteria, and hence there is a need for new technologies that can quantify antibiotic transport across biological membranes. We recently developed an optofluidic assay that directly determines the permeability coefficient of autofluorescent drug molecules across lipid membranes. Using ultraviolet fluorescence microscopy, we directly track drug accumulation in giant lipid vesicles as they traverse a microfluidic device while exposed to the drug. Importantly, our measurement does not require the knowledge of the octanol partition coefficient of the drug - we directly determine the permeability coefficient for the specific drug-lipid system. In this work, we report measurements on a range of fluoroquinolone antibiotics and find that their pH dependent lipid permeability can span over two orders of magnitude. We describe various technical improvements for our assay, and provide a new graphical user interface for data analysis to make the technology easier to use for the wider community.